Polyketides are a diverse class of naturally occurring molecules typically produced by a variety of organisms, including fungi and mycelial bacteria, in particular actinomycetes. Although polyketides have widely divergent structures, they are classified together because they all share a common general biosynthetic pathway in which the carbon backbone of these molecules are assembled by sequential, step-wise addition of two carbon or substituted two carbon units referred to as ketides. Polyene polyketides comprise a chain of ketide units that have been strung together by a series of enzymatic reactions by multimodular polyketide synthase proteins.
Polyketides are usually found in their natural environment in trace amounts. Moreover, due to their structural complexity, poyketides are notoriously difficult to synthesize chemically. Nevertheless, many polyketides have been developed into effective drugs for the treatment of conditions such as bacterial and fungal infections, cancer and high cholesterol. Adriamycin, erythromycin, zocor and nystatin are but a few examples of polyketide molecules, which have been developed into valuable pharmaceuticals. Linearmycin A, having a 60 carbon chain and a degree of unsaturation of 15, is an example of a linear polyene polyketide reported to possess antifungal and antibacterial activity (Sakuda et al., Tetrahedron Letters. Vol. 36, No. 16, 2777-2870 (1995); Sakuda et al., J. Chem Soc., Perkin Trans. 1,2315-2319 (1996)).
Although large numbers of therapeutically important polyketides have been identified, there remains a need to obtain novel polyketides that have enhanced properties or possess completely novel bioactivities. The complex polyketides produced by modular Type I polyketide synthases (PKSs) are particularly valuable, in that they include compounds with known utility as antihelminthics, insecticides, immunosuppressants, cytotoxic, antifungal or antibacterial agents.
Because of their structural complexity, such novel polyketides are not readily obtainable by total chemical synthesis. The present invention addresses this need by providing a new class of polyketide compounds with therapeutic activity, together with means for their production. The compounds of the invention are prepared by fermentation or by fermentation followed by chemical modifications. The compounds of the invention may also be produced by appropriate application of recombinant DNA technology. A wide variety of polyketides can be prepared in a variety of different host cells provided one has access to nucleic acid compounds that encode PKS proteins and polyketide modification enzymes.
PKSs are large proteins that contain multiple enzymatic activities. PKSs catalyse the biosynthesis of polyketides through repeated, decarboxylative Claisen condensations between acylthioester building blocks, such as acetyl, butyryl, isobutyryl, propionyl, malonyl, hydroxymalonyl, methylmalonyl, and ethylmalonyl CoA.
PKS enzymes are generally classified into Type I or “modular” PKSs and Type II or “iterative” PKSs according to the polyketide synthetized and by the mode of synthesis. Type I PKSs are responsible for producing a large number of 12-, 14- and 16-membered macrolide antibiotics.
Type I or modular PKS enzymes are multifunctional proteins containing catalytic sites for acyl transferases (AT), acyl carrier protein (ACP), ketosynthase (KS), dehydratase (DH), and enoyl reductase (ER) activities. Type I enzymes are formed by a set of separate catalytic active sites for each cycle of carbon chain elongation and modification in the polyketide synthesis pathway. Each active site is termed a domain. A set of active sites or domains is termed a module. The typical modular PKS complex is composed of several large PKS polypeptides that act coordinately to achieve polyketide synthesis. Each PKS polypeptide can be segregated from amino to carboxy terminus into a loading module (found only in the first PKS polypeptide of the complex), multiple extender modules, and a releasing or thioesterase (TE) domain (generally found only in the final module of the terminal PKS polypeptide of the complex).
Generally, the loading module is responsible for binding the first building block used to synthesize the polyketide and transferring it to the first extender module. The AT domain of the loading module recognizes a particular acyl-CoA (usually acetyl or propionyl but sometimes butyryl, isobutyryl or other acyl-CoA) and transfers it as a thiol ester to the ACP domain of the loading module. The loading module may not encode a KS domain, or may encode a KS(Q) domain, a KS-like domain that carries an amino acid substitution at the active site cysteine residue (typically a glutamine residue, single letter code Q). KS(Q) domains decarboxylate the acylthioester of the loading domain before proceeding with chain elongation. For example, the loader module of the oleandomycin PKS complex initiates deoxyoleandolide synthesis by loading the ACP with a malonyl unit and performing a decarboxylation to generate acetyl-ACP (Shah, (2000), J. Antibiotics, Vol. 53, pp. 502-508).
The AT domain on each of the extender modules recognizes a particular extender-CoA (typically malonyl or alpha-substituted malonyl, i.e. methylmalonyl, ethylmalonyl, and 2-hydroxymalonyl) and transfers it to the ACP domain of that extender module to form a thioester. Each extender module is responsible for accepting a compound from a prior module, binding a building block, attaching the building block to the compound from the prior module, optionally performing one or more additional functions, and transferring the resulting compound to the next module.
Each extender module of a modular PKS contains a KS, AT, ACP, and zero, one, two or three domains that modify the beta-carbon of the growing polyketide chain. A typical (non-loading) minimal Type I PKS extender module may contain a KS domain, an AT domain, and an ACP domain. Such domains are sufficient to activate a 2-carbon extender unit and attach it to the growing polyketide molecule. The next extender module, in turn, is responsible for attaching the next building block and transferring the growing compound to the next extender module until synthesis of the polyketide is complete.
Once the PKS is primed with acyl- and malonyl-ACPs, the acyl group of the loading module is transferred to form a thiol ester (trans-esterification) at the KS of the first extender module; at this stage, extender module one possesses an acyl-KS and a malonyl (or substituted malonyl)-ACP. The acyl group derived from the loading module is then covalently attached to the alpha-carbon of the malonyl group to form a carbon-carbon bond, driven by concomitant decarboxylation, and generating a new acyl-ACP that carries a backbone two carbons longer than the loading building block (elongation or extension) and side chains if a substituted malonyl unit is used for extension.
The polyketide chain, growing by two or more carbons with each extender module, is sequentially passed as covalently bound thiol esters from extender module to extender module, in an assembly line-like process. The carbon chain produced by this process alone would possess a ketone at every other carbon atom, producing a polyketone, from which the name polyketide arises. Most commonly, however, additional enzymatic activities modify the beta keto group of each two carbon unit just after it has been added to the growing polyketide chain but before it is transferred to the next module.
After traversing the final extender module, the polyketide encounters a releasing domain (TE) that cleaves the polyketide from the PKS and typically cyclizes the polyketide. Further, tailoring enzymes can modify the polyketide; these tailoring enzymes add carbohydrate groups, methyl groups, or make other modifications, i.e. oxidation or reduction, on the polyketide core molecule.
Type I PKSs displays a one-to-one correlation between the number and clustering of active sites in the primary sequence of the PKS and the structure of the polyketide backbone. The activities catalyzed by the domains within a type I PKS are often apparent in the structure of the growing polyketide chain; consequently, nucleotide sequence has become a predictive tool for deducing the biosynthetic route for these compounds (Rangaswamy et al, Proc. Natl. Acad. Sci. USA, (1998) Vol. 95, pp. 15469-15474).
In Type I PKS polypeptides, the order of catalytic domains is conserved. When all beta-keto processing domains are present in a module, the order of domains in that module from N-to-C-terminus is always KS, AT, DH, ER, KR, and ACP. Some or all of the beta-keto processing domains may be missing in particular modules, but the order of the domains present in a module remains the same. The order of domains within modules is believed to be important for proper folding of the PKS polypeptides into an active complex. Importantly, there is considerable flexibility in PKS enzymes, this flexibility provides a means for genetically engineering novel catalytic complexes. By manipulating the polynucleotide sequences encoding the PKS polypeptide, genetically engineered novel PKSs can be achieved. Genetically engineering PKS enzymes can be achieved by the modification, addition or deletion of domains, or by replacing domains with domains taken from other Type I PKS enzymes. As well, this can also be achieved by deletion, addition or replacement of entire modules with modules taken from other sources. A genetically engineered PKS complex should, of course, have the ability to catalyze the synthesis of the product predicted from the genetic alterations made. Alignment of the many available amino acid sequences for Type I PKS enzymes has approximately defined the boundaries of the various catalytic domains. Sequence alignments also have revealed linker regions between the catalytic domains and at the N- and C-termini of individual PKS polypeptides. The sequences of these linker regions are less well conserved than are those for the catalytic domains, which is in part how linker regions are identified. Linker regions can be important for proper association between domains and between the individual polypeptides that comprise the PKS complex. One can thus view the linkers and domains together as creating a scaffold on which the domains and modules are positioned in the correct orientation to be active. This organization and positioning, if retained, permits PKS domains of different or identical substrate specificities to be substituted (usually at the DNA level) between PKS enzymes by various available methodologies. In selecting the boundaries of, for example, an AT domain replacement, one can thus make the replacement so as to retain the linkers of the recipient PKS or to replace them with the linkers of the donor PKS AT domain, or, preferably, make both constructs to ensure that the correct linker regions between the KS and AT domains have been included in at least one of the engineering enzymes. Thus, there is considerable flexibility in the design of new PKS enzymes with the result that known polyketides can be produced more effectively, and novel polyketides can be made.